FIG. 5.
Endothelial attachment was compared at 1 h between L-MSCs and BM-MSCs. (A) Significantly more L-MSCs than BM-MSCs attached to human umbilical vein endothelial cells (HUVECs) (*P<0.05). Anti-ICAM-1 blocking antibody (clone YN1/1.7.4, 10 μg/mL) significantly reduced the adherence of L-MSCs to HUVEC relative to treatment with isotype Ab. Phase-contrast images (100×) of study showing adherence of L-MSCs or BM-MSCs against darker background of HUVEC monolayer (lower panel). Higher magnification (400×) images showing spreading of adherent L-MSCs (inset). (B) Endothelial adherence study employing PKH-labeled BM-MSCs or L-MSCs and analyzed on the basis of total fluorescence. Group data showing statistically significant increased total pixel density of adherent L-MSCs incubated with isotype or no antibody versus BM-MSCs incubated with isotype antibody (rat IgG2b) and a significant reduction in attachment and spreading of L-MSCs when incubated (1 h) with anti-ICAM-1 functional blocking Ab (†P<0.05 vs. isotype Ab). (C) Blocking Ab effect on attachment of L-MSCs to endothelial monolayers was also concentration dependent (ICAM-1 group, *P<0.05, 10 vs. 1 μg/mL). (D) Association between ICAM-1 expression and lung retention of L-MSCs in elastase-injured mice. Explant-derived L-MSCs with high (87.8%) versus low (23.0%) incidence of ICAM-1 expression were generated by repeated passage using trypsin-free reagent (TrypLE; Invitrogen) versus trypsin/ethylenediaminetetraacetic acid, respectively. Passage 7 high versus low ICAM-1 expressing L-MSCs (0.5×106/mouse) were injected intravenously and retention quantified 4 days after treatment (n=5 mice/group) using flow cytometry. Representative flow cytometry plots from mice injected with high, low, or no L-MSCs injected showed significantly higher retention of high ICAM-1 versus low ICAM-1 expressing L-MSCs. (E) High ICAM-1 expression resulted in significantly (*P=0.003) greater retention efficiency (% total cells/injected cells in millions) and (F) Lower phagocytosis rates by total leukocytes or macrophages. Color images available online at www.liebertonline.com/scd